Use of peptide vectors to improve the immune response to antigens

ABSTRACT

The invention relates to conjugates of an antigen coupled to a linear derivative of a β-stranded antibiotic peptide, which are useful for immunogenic agents to enhance a CTL response. Two groups of preferred peptides are derived from the antibiotics protegrin and tachyplesin.

FIELD OF INVENTION

[0001] The present invention relates generally to the field ofimmunology and vaccine technology. More specifically, the presentinvention relates to methods of delivering antigens into cells in orderto enhance their immune response for use in vaccination and prophylaxis.

BACKGROUND OF THE INVENTION

[0002] The host immune system provides a sophisticated defence mechanismthat enables the recognition and elimination of foreign entities, suchas infections agents or neoplasms, from the body. When functioningproperly, an effective immune system distinguishes between foreigninvaders and the host's own tissues. The first response to foreignagents is the secretion of antibodies that are able to recognize, blockand destroy microbial agents. However, this response is often notsufficient because in some cases, such as viral particles, the pathogensare able to escape B cell antibody response by rapidly entering intotarget cells where the antibodies cannot reach them. The pathogen canthen replicate intracellularly and infect other peripheral cells. Thechallenge for scientists is to enhance the T-cell response againstmicrobial agents. T-cell response is the capacity of the immune systemto raise a special type of lymphocytes [CD4+, CD8+] that are able torecognize specifically the infected cells and destroy them. Thismechanism of T-cell response is complementary to the B-cell antibodyresponse and both are needed to elicit an efficient immune response. Asan example, an efficient vaccine against HIV infection is a long processbecause of the difficulty to generate a CTL response against variousvaccine candidates.

[0003] Dendritic cells (DCs) are efficient antigen presenting cells(APC) that initiate immune response to peptide antigens associated withclass I and II MHC (Freudenthal, P. S. and Steinman, R. M., Proc. Natl.Acad. Sci. USA 87:7698, 1990; Steinman, R. M., Ann. Rev. Immune. 9:271,1991). DCs represent a small subpopulation of widely distributed, bonemarrow-derived leucocytes, which are the only natural antigen presentingcells able to prime naive T cells. They activate both CD4+ and CD8+ Tlymphocyte primary immune response, and are at least as effective asother APCs such as monocytes in stimulating secondary immune responses(Peters et al., Immunol. Today L7:273, 1997).

[0004] In order to stimulate T lymphocyte responses, peptide fragmentsfrom antigens contained in a vaccine must first be bound to peptidebinding receptors (major histocompatibility complex [MHC] class I and IImolecules) that display the antigenic peptides on the surface of antigenpresenting cells (APCs). T lymphocytes produce an antigen receptor thatthey use to monitor the surface of APCs for the presence of foreignpeptides. Current models of antigen processing and presentation to Tlymphocytes suggest that two principle pathways exist. In brief,exogenous antigens are internalised into the endocytic compartments ofAPCs where they are hydrolysed into peptides, some of which become boundto MHC class II molecules. The mature MHC class II/peptide complexes arethen transported to the cell surface for presentation to classII-restricted CD4⁺ T lymphocytes. In contrast, for the MHC class Imolecules, endogenous antigens are degraded in the cytoplasm by theaction of a proteolytically active particle known as the proteasomebefore their transport into the endoplasmic reticulum, where they bindto nascent MHC class I molecules. Stable class I/peptide complexes aretransported through Golgi apparatus to the cell surface to CD8⁺ CTL.Because the CTL response is crucial for protection against many viral orparasitic infections and some tumour cells, several new vaccinestrategies have been proposed: 1) Immunostimulating complexes (Takahasciet al. 1990. Nature 344:873); 2) antigen-loaded pH-sensitive liposomes(Nair et al. 1992. J. Exp. Med. 175:609); 3) recombinant bacteriaexpressing foreign antigens (Tuner et al. 1993, Infect. Immun. 61:5374;Ikonomidis et al. 1994. J. Exp. Med 180:2209); 4) bacterial toxins fusedto CTL epitopes (Donnelly et al. 1993. Proc; Natl. Acad. Sci USA90:3530); 5) particulate antigens (Schirmbeck et al. 1994. Eur. J.Immunol 24:2068, Layton et al, 1993. J. Immunol 151:1097); 6) use ofvarious vectors (Schutze-Redelmeier et al. 1996, J. Immunology157:650-655; Schluesener 1996, J Neurosci Res 46:258-262); and 7) nakedDNA injected in muscle cells (Ulmer et al. 1993. Science 259:1745). Thisvariety of strategies reflects the inherent difficulty of deliveringantigens intracellularly in order to elicit a CTL response. In manycases, these approaches have a poor in vivo efficiency and are limitedby safety considerations, immune responses against the vector, and cost.

[0005] In addition to the immune system, mammals are known to producesmall peptides which have direct antimicrobial activity. Most of thesepeptides act by causing direct lysis of the membrane of prokaryotes. Amajor family of these peptides are β-stranded antibiotic peptides linkedby disulphide bonds. Members of the family include defensins (Lehrer etal, 1991, Cell 64:229-230; Lehrer et al, 1993, Ann. Rev. Immunol.11:105-128), protegrins (Kokryakov et al, 1993, FEBS 337:231-236) andtachyplesins (Nakamura et al, 1988, J. Biol. Chem. 236:16709-16713;Miyata et al, 1989, J. Biochem. 106:663-668).

[0006] Peptides of these classes are known to be able to pass throughthe membranes of mammalian cells, though due to the differences betweenbacterial and mammalian cell membranes, the peptides are non-toxic tomammalian cells.

[0007] WO99/07728 describes a number of derivatives of these peptides asvectors for the introduction of substances to cells or for substances topass through the blood-brain barrier. These derivatives include linearderivatives in which the peptides do not have disulphide bonds. Theabsence of disulphide bonds is brought about by substitution of cysteineresidues, or blocking their terminal thiol groups.

DISCLOSURE OF THE INVENTION

[0008] The applicants have surprisingly found that when peptide vectorsbased upon the peptides of WO99/07728 are used to attach an antigen, theresulting product can be taken up by antigen presenting cells which arethen able to process the antigen and display the antigen on its surfacein a manner to facilitate a CTL response. It has been found that the CTLresponse is enhanced in comparison to the use of the antigen alone.

[0009] In a first aspect, the invention provides a conjugate of anantigen coupled to a linear derivative of a β-stranded antibioticpeptide. Preferably, these linear derivatives do not exhibitantibacterial activity.

[0010] The invention also provides a pharmaceutical compositioncomprising said conjugate in a pharmaceutically acceptable carrier.

[0011] In another aspect, the invention provides a method of enhancingan immune response to an antigen in a mammal, said method comprisingadministering to the mammal an effective amount of a conjugate of saidantigen coupled to a linear derivative of a β-stranded antibioticpeptide. The invention also provides the use of a conjugate of anantigen coupled to a linear derivative of a β-stranded antibioticpeptide for the manufacture of a medicament for enhancing an immuneresponse to said antigen in a mammal.

[0012] The invention also provides a conjugate of an antigen coupled toa linear derivative of a β-stranded antibiotic peptide for use in amethod of enhancing an immune response to said antigen in a mammal.

DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 shows the uptake of conjugates SynB3-fluNP and SynB4-fluNPby K562 cells, together with a control fluNP peptide. The x axis is timein minutes and the y axis denotes Mean Fluorescence Intensity.

[0014]FIG. 2 shows the CTL responses elicited from mice immunized withcontrols ((a), (b) and (c)) and conjugates of the invention ((d) and(e)).

[0015]FIG. 3 shows the uptake of conjugates SynB3-DPV and SynB4-DPV byK562 cells, together with a control DPV protein. The x axis is time inminutes and the y axis denotes Mean Fluorescence Intensity.

[0016]FIG. 4 shows CTL responses elicited from mice immunized withcontrols and DPV conjugates of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Linear Derivative of a β-Stranded Antibiotic Peptide.

[0018] This term refers to any mammalian β-stranded antibiotic peptidewhich has been modified to remove internal disulphide bonds formedbetween cysteine residues, and optionally further modified bysubstitution, insertion or deletion in a manner which retains theability of the peptide to cross the membrane of a mammalian cell.

[0019] Modification to remove disulphide bonds may be achieved bychemically blocking the thiol group (e.g. by conversion to an R-thiogroup such as methyl-thio), or by substitution of the cysteine residueby another amino acid, such as serine, alanine or glycine.

[0020] Since a disulphide bond requires two cysteine residues, it is ofcourse possible to leave one unmodified residue in the peptide though isthis not preferred, so as to avoid dimers forming.

[0021] In one aspect, the linear peptide may be defined as a peptide ofstructure: Nter-Mid-Cter, in which Mid is a peptide of formula (I):

X1/3-(X1/2 or a bond)-(X or a bond)-X3-(X1 or a bond)-X1-X-X1/2-(X2/3 ora bond)-Db-(X2/3 or a bond)-X1/3  (SEQ ID NO:1);

[0022] where Db is either X3-X3 or X1-X1; and

[0023] wherein Nter is either an N-terminus or

X1/X3-X1-X1/2-X1-X3  (SEQ ID NO:2); and

[0024] wherein Cter is either a C-terminus or

X1/2-X1/2-X2/3-X1/2-X1-X3  (SEQ ID NO:3).

[0025] in which:

[0026] each X1, which may be identical or different, represents an aminoacid residue for which the side chain is non-polar;

[0027] each X2, which may be identical or different, represents an aminoacid residue for which the side chain is polar; and

[0028] each X3, which may be identical or different, represents an aminoacid residue for which the side chain is basic;

[0029] X is any one of X1, X2 and X3; wherein said peptide is linear(i.e. does not contain any intra-molecular disulphide bonds) and retainsthe ability to cross a mammalian membrane; or a fragment thereofretaining the ability to cross a mammalian membrane.

[0030] In the above peptide, it is preferred that if the value indicatedas “X1 or a bond” is a bond, then there is a residue X2 or X3 at theposition indicated as the first (from the N- to C terminal direction)occurrence of “X2/3 or a bond”. Likewise, where the latter is a bond, itis preferred that the former is X1.

[0031] Amino acids which have non-polar side chains include alanine,glycine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, cysteine, norleucine, cysteine^(ACm),penicillamine, proline, norvaline, phenylglycine, Abu, carboxylicamino-1-cyclohexane acid, Aib, carboxylic 2-aminotetraline,4-bromophenylalanine, tert-Leucine, 4-chlorophenylalanine,3,4-dichlorophenylalanine, 4-fluorophenylalanine, homoleucine,4-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine,4-nitrophenylalanine, 3-nitrotyrosine, 3-pyridylalanine,[2-thienyl]alanine.

[0032] Preferred non-polar side chain amino acids are glycine, valine,norvaline, leucine, isoleucine, norleucine, proline, phenylalanine,methionine and tryptophan.

[0033] Amino acids which have a polar side chain include serine,threonine, tyrosine, asparagines, glutamine, citrulline, homocitrulline,isoasparagine, β-homoglutamine, β-homoglutamine β-glutamine,β-homoserine, β-homothreonine, homoserine, isoserine.

[0034] Preferred such amino acids are serine, threonine, tyrosine,asparagines and glutamine.

[0035] Amino acids with a basic side chain include arginine, lysine,histidine, ornithine, diaminoacetic acid, diaminobutyric acid, anddiaminopropionic acid. Preferred such amino acids include arginine andlysine. Arginine is particularly preferred.

[0036] Thus in a preferred embodiment, X1 is selected from glycine,valine, norvaline, leucine, isoleucine, norleucine, proline,phenylalanine, methionine and tryptophan, X2 is selected from serine,threonine, tyrosine, asparagines and glutamine and X3 is selected fromarginine and lysine.

[0037] In a particular subclass of the above formula, the peptide may bea protegrin derivative of formula:

R-(Xa)-R-L-X1/2-Y-X2/3-Db-(R or a bond)-F-X1/2-X1/2-X2/3-X1/2-X1-R  (SEQID NO:4)

[0038] where Xa is either X1-X1 or a bond, or a fragment thereof of atleast 7 amino acids.

[0039] Preferably where Xa is X1-X1, the two groups are the same.

[0040] More preferably, the formula is:

R-(Xa)-R-L-(G/S/A)-Y-(R/S)-Db-R-F-(G/S/A)-X1-(R/S)-(V/T)-G-R  (SEQ IDNO:5);

[0041] and most preferably the formula is:

R-(Xb)-R-L-(G/S/A)-Y-(R/S)-R-R-R-F-(G/S/A)-(T/I/V)-(R/S)-(V/T)-G-R  (SEQID NO:6)

[0042] where Xb is either a bond or A-A or G-G.

[0043] Examples of peptides of the above formula include:

R-G-G-R-L-S-Y-S-R-R-R-F-S-V-S-V-G-R  (SEQ ID NO:7)

R-A-A-R-L-A-Y-R-L-L-R-F-A-I-R-V-G-R  (SEQ ID NO:8)

R-A-A-R-L-G-Y-R-_(n)L-_(n)L-R-F-G-Z-R-V-G-R  (SEQ ID NO:9)

R-G-G-R-L-S-Y-S-R-R-R-F-S-T-S-T-G-R  (SEQ ID NO:10)

R-R-L-S-Y-S-R-R-R-F  (SEQ ID NO:11)

[0044] in which _(n)L is norleucine, and Z is norvaline.

[0045] In another preferred embodiment, the peptide is a tachyplesinderivative of formula:

X1/X3-X1-X1/2-X1-R-X1-X1/2-X2-R-X1-X1-S/R-X2-Db-X2/3-X2/3  (SEQ IDNO:12);

[0046] or a fragment thereof of at least 7 amino acids.

[0047] Preferably the two residues of Db are the same as each other.

[0048] Preferably this formula is:

(K/R/A)-W-(S/A)-F-R-X1-(S/A)-Y-R-X1-X1-(S/R)-Y-Db′-(R/S)-(R/L/_(L))  (SEQID NO:13)

[0049] where Db′ is selected from L-L, _(n)L-_(n)L and R-R (where _(n)Lis norleucine).

[0050] More preferably, the formula is:

(K/R/A)-W-(S/A)-F-R-V-(S/A)-Y-R-G-I-(S/R)-Y-R-R-R-(R/L)  (SEQ ID NO:14).

[0051] Examples of peptides include:

K-W-S-F-R-V-S-Y-R-G-I-S-Y-R-R-S-R  (SEQ ID NO:15);

R-W-S-F-R-V-S-Y-R-G-I-S-Y-R-R-S-R  (SEQ ID NO:16);

K-W-A-F-R-V-A-Y-R-G-I-R-Y-L-L-R-L  (SEQ ID NO:17); and

A-W-S-F-R-V-S-Y-R-G-I-S-Y-R-R-S-R  (SEQ ID NO:18).

[0052] For the avoidance of doubt, in the above peptide formulae, thestandard 1-letter amino acid code is used to represent the naturallyoccurring amino acids. Other letters are used as defined. The presenceof alternative residues at one position is indicated as “/”; thus X1/2means the residue in the peptide may be either of X1 or X2, andsimilarly R/S means that arginine or serine may occur where indicated.

[0053] All the peptides of the invention may comprise amino acids in theL-configuration or in the D-configuration. Further, these L- orD-peptides may be in the retroform, i.e. sequences in which the N- toC-terminal order is reversed. An example of such a peptide is:

R-S-R-R-Y-S-I-G-R-Y-S-V-R-F-S-W-A  (SEQ ID NO:19),

[0054] which is the retro form of the first tachyplesin-derived peptideshown in the preceding paragraph.

[0055] Fragments

[0056] Fragments of the above sequences which retain the ability of thepeptide to cross a mammalian membrane may also be used. Generally, thefragments will be at least 7 amino acids in size. Preferably thepeptides will be in the size range of 7 to 24 amino acids, such as 10 to24, e.g. 10 to 20 in size. A typical size range within this is from 12to 20 amino acids in size.

[0057] Basic Residues

[0058] Peptides and fragments of the invention contain a number of aminoacids X3, which are preferably arginine or lysine. It is preferred thatpeptides and fragments are selected to contain at least 4, preferably atleast 5, and more preferably at least 6 residues X3.

[0059] N- and C-Terminal Extensions of the Peptides

[0060] Peptides (including fragments) of the invention will function asa vector for the enhancement of an immune response to an antigen. Wherethe antigen is attached to the N- or C-terminus of the peptide, theantigen may be attached directly by an amide bond, or indirectly via alinker. In the case of the latter, the linker may be from 1 to 25 aminoacids and composed of any suitable peptide sequence. The linker may be aflexible linker of the type used to link antibody heavy and light chainstogether in a single chain antibody.

[0061] Where the N- or C-terminal of the peptide vector is not attachedto the antigen, it may optionally contain a short amount of additionalsequences, for example from 1 to 25 residues, which may for example bepresent for reasons conventional in the art of genetic engineering. Forexample, the sequence may form a short tag for purification oridentification purposes, or may form a cleavable pre-sequence fortransport out of a host cell in which the peptide-antigen fusion isproduced.

[0062] Where the N-terminal of the peptide is not extended by thepresence of additional sequences or is not linked to the antigen, theN-terminal of the peptide will generally comprise an amino group, thoughmodifications to the group, such as those which may result from chemicalsynthesis of the peptide, may be present. Likewise, the C-terminal of apeptide may be a modified carboxy terminal, such as an amidated carboxygroup or the like.

[0063] Retains the Ability to Cross a Mammalian Membrane.

[0064] By this term, it is meant that the peptide will be able to crossthe cell membrane of a mammalian cell in culture at 37° C. to at least50%, preferably at least 75% of the penetration achieved by the peptideSynB3 at a concentration of (both of) 1 μM, measured after 60 minutes inculture. The mammalian cell may be a primary cell line, a cancer cellline or any cell line generally available in the art, such as a K562cells.

[0065] Antigen.

[0066] The antigen coupled to the peptide may be any antigen to which itis desired to provoke an immune response in a host mammal.

[0067] Antigens include peptides, whole proteins, and protein subunits.

[0068] The antigens may be viral, bacterial or derived from autologousproteins, for example for use in the treatment of autoimmune diseases orcancers.

[0069] Exemplary viral antigens include, but are not limited to,antigens derived from influenza virus; adenovirus; hepatitis A, B and Cviruses; yellow fever virus; dengue fever virus; HIV-1 and HIV-2; HSV1and HSV2; Epstein-Barr virus; Retroperitoneal fibromatosis associatedherpes virus, Human papilloma virus, Kaposi's sarcoma herpes virus, andcytomegalovirus (CMV).

[0070] Exemplary bacterial antigens include, but are not limited to,antigens from infectious bacteria such as Mycobacterium tuberculosis,Acne vulgaris, Propionibacterium acnes, Chlamydia trachomatis, Babesiamicroti, Ehrlichia risticii, Borrelia burgdorferi, Leishmaniaaethiopica, Candida albicans, Mycobacterium tuberculosis, Staphylococcusaureus, Staphylococcus pyogenes, Staphylococcus epidermis,Staphylococcus sapropyticus, and Trypanosoma cruzi.

[0071] Self-antigens include antigens that appear on cells associatedwith the onset of autoimmune diseases or cancer. Exemplary antigens areassociated with the following cancers: Acute myelogenous leukaemia(AML), Acute lymphocytic leukaemia (ALL), Chronic myelogenous leukaemia(CML), Chronic lymphocytic leukemia (CLL), Hairy cell leukemia, Myeloma,and all solid tumors of all tissue types.

[0072] The accompanying examples illustrate the use of a peptide antigenof just 9 amino acids, and a 8.4 kDa protein derived from M.tuberculosis. Accordingly, the peptide vector may be used with a widerange of antigens, e.g. from a single peptide epitope of about 6 aminoacids to proteins or subunits thereof of at least 200 kDa, thoughpreferably no more than 100 Kda.

[0073] Antigens also include DNA or oligonucleotides that can be usedfor DNA-based vaccination where immunogenic proteins are expressed in invivo transfected cells of the vaccine recipients in their nativeconformation from antigen-encoding expression plasmid DNA.

[0074] Mammal.

[0075] By “mammal”, this is intended to be any mammal, including ahuman. Conjugates of the invention may be useful in veterinary medicine,e.g. for the vaccination of livestock and poultry or pets, as well as inhuman medicine.

[0076] Enhancing the Immune Response.

[0077] Conjugates of the invention will be useful in provoking an immuneresponse in a subject mammal which is greater than the immune responsewhich would be achieved in the mammal by the administration of an amountof unconjugated antigen equivalent to the amount of antigen in theconjugate. The ability of a conjugate to do this may be measured in anumber of ways known in the art. An assay to measure CTL response inmice, illustrated in the accompanying examples, is one such method.

[0078] Preparation of Conjugates.

[0079] Conjugates may be prepared by chemical synthesis or by usingmolecular biology techniques. The antigen substance may be coupled to apeptide vector in the compositions according to the invention by anyacceptable bonding means considering the chemical nature, the size andnumber of active and associated substances and peptides. They may becovalent, hydrophobic or ionic bonds, or cleavable or non-cleavablebounds in the physiological media or inside cells.

[0080] Coupling may be achieved in any site in the peptide vector inwhich functional groups such as —OH, —SH, —COOH, —NH₂ are naturallypresent or have been introduced. Thus an antigen molecule may be coupledto the peptide at the N-terminal or C-terminal ends, or in the peptideside chains.

[0081] Similarly, coupling may be achieved on any site in the antigenmolecule, for example at which functional groups such as —OH, —SH,—COOH, —NH₂ are naturally present or have been introduced.

[0082] Coupling of the antigen may also occur by non-covalent means. Forexample, the vector peptide may comprise a group (e.g. streptavidin)which binds to a cognate group attached to the antigen (e.g. biotin).Ionic groups attached to the peptide vector and antigen may also providesuitable coupling. The linker may be designed to be cleavable within anAPC, so as to facilitate the processing and presentation of the antigen.For example, a disulfide link may be used since these linkers aregenerally stable in plasma and reduced in the cell.

[0083] It is possible to couple more than one antigen to each vectorpeptide, and/or vice versa. This will depend to some extent on therelative sizes of the vector and antigen. Thus the ratio of peptidevector molecules to antigen molecules per conjugate may vary from 10:1to 1:10, preferably from 5:1 to 1:5.

[0084] Where the peptide vector and antigen are joined by C- toN-terminal fusion (in either order), the fusion may be prepared as asingle fusion protein by recombinant means.

[0085] Accordingly, another aspect of the invention is a nucleic acidmolecule encoding such a fusion. The nucleic acid may be DNAs or RNAsand may be associated with control sequences such as a promoter and/orinserted in vectors. The vector used is chosen to be compatible with thehost into which it will be transferred, to provide for expression of thefusion protein. Preparation of these vectors, and production orexpression of peptides or compounds with a type (II) formula in a host,may be produced using molecular biology and genetic engineeringtechniques well known to those skilled in the art.

[0086] Thus in another embodiment, the invention provides a method ofmaking a conjugate as defined above, which method comprises expressingin a host cell culture a nucleic acid sequence encoding said conjugateand recovering said conjugate from the culture.

[0087] Compositions.

[0088] Compositions of the invention comprise conjugates of theinvention and a pharmaceutically acceptable carrier. Compositions may beformulated for any suitable route and means of administration.Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral, rectal, nasal, topical (including buccaland sublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general the formulations are preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product.

[0089] For solid compositions, conventional non-toxic solid carriersinclude, for example, pharmaceutical grades of mannitol, lactose,cellulose, cellulose derivatives, starch, magnesium stearate, sodiumsaccharin, talcum, glucose, sucrose, magnesium carbonate, and the likemay be used. The active compound as defined above may be formulated assuppositories using, for example, polyalkylene glycols, acetylatedtriglycerides and the like, as the carrier. Liquid pharmaceuticallyadministrable compositions can, for example, be prepared by dissolving,dispersing, etc, an active compound as defined above and optionalpharmaceutical adjuvants in a carrier, such as, for example, water,saline aqueous dextrose, glycerol, ethanol, and the like, to therebyform a solution or suspension. If desired, the pharmaceuticalcomposition to be administered may also contain minor amounts ofnon-toxic auxiliary substances such as wetting or emulsifying agents, pHbuffering agents and the like, for example, sodium acetate, sorbitanmonolaurate, triethanolamine sodium acetate, sorbitan monolaurate,triethanolamine oleate, etc. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 15th Edition, 1975. The composition or formulationto be administered will, in any event, contain a quantity of the activecompound(s) in an amount effective to alleviate the symptoms of thesubject being treated.

[0090] Dosage forms or compositions containing active ingredient in therange of 0.25 to 95% with the balance made up from non-toxic carrier maybe prepared.

[0091] For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example, pharmaceutical grades ofmannitol, lactose, cellulose, cellulose derivatives, sodiumcrosscarmellose, starch, magnesium stearate, sodium saccharin, talcum,glucose, sucrose, magnesium, carbonate, and the like. Such compositionstake the form of solutions, suspensions, tablets, pills, capsules,powders, sustained release formulations and the like. Such compositionsmay contain 1%-95% active ingredient, more preferably 2-50%, mostpreferably 5-8%.

[0092] Parenteral administration is generally characterized byinjection, either subcutaneously, intramuscularly or intravenously.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate, triethanolamine sodium acetate, etc.

[0093] A more recently devised approach for parenteral administrationemploys the implantation of a slow-release or sustained-release system,such that a constant level of dosage is maintained. See, e.g., U.S. Pat.No. 3,710,795.

[0094] The percentage of active compound contained in such parentalcompositions is highly dependent on the specific nature thereof, as wellas the activity of the compound and the needs of the subject. However,percentages of active ingredient of 0.1% to 10% in solution areemployable, and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. Preferably, thecomposition will comprise 0.2-2% of the active agent in solution.

[0095] Doses and Routes of Administration.

[0096] The conjugate of the invention, for example in the form of acomposition discussed above, may be administered by different pathways,for example by oral, rectal, nasal, topical (including buccal andsublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural)administration.

[0097] The effective amount of the conjugate of the invention to beadministered will ultimately be at the discretion of the physician,taking into account the severity of the disease in a particular subject(e.g. a human patient or animal model) and the overall condition of thesubject. Suitable doses will typically be in the range of 1 μg to 10 mgof antigen, more preferably between 10 μg and 1 mg of antigen, and stillmore preferably between 100 μg and 1 mg of antigen.

[0098] In order to induce an immune response in a mammalian subject, theconjugate may be administered in repeat doses, e.g. to provide boosterdoses at repeat intervals.

[0099] The conjugate may be used to boost the immunity of patientshaving a particular condition or to provide an immune response to aparticular condition. Preferably, the conjugate provides a protectiveimmune response to the condition.

[0100] Other advantages and characteristics of the invention will becomeclear after reading the following example concerning the preparation ofcompounds with a type (II) formula in which a peptide epitope and aprotein antigens have been coupled to peptide vectors, and theirenhancement of cell uptake and CTL response according to the invention.

EXAMPLES

[0101] Protocols

[0102] Peptide Synthesis

[0103] All peptides were synthesized according to Fmoc-tBu strategyusing an AMS 422 (ABIMED, Germany). Labelling of the N-terminus of thepeptides with NBD probe (4-fluoro-7-nitrobenzofurazan) was achieved asdescribed elsewhere (Gazit et al., 1995 Biochemistry 34:11479-11488).Peptide purification was accomplished by reverse phase HPLC.Purification was over 95% for all peptides by the criterion of UVabsorbance at 220 nm and 460 nm.

[0104] Coupling of SynB Vectors to the Recombinant Protein rDPV

[0105] The N-terminal amine of the rDPV protein was first derivatisedwith 2-Iminithiolane (2IT) in order to have a free thiol, whilepreserving the positive charge. Excess 2-IT reagent was removed from theprotein by gel filtration. The 2IT-rDPV was then incubated with athiopyridinium-activated 3MP-SynB peptide, to allow the formation of adisulfide bridge, by displacement of thiopyridinium from the 3MP-Peptideby the free thiol of 2IT-DPV. The displaced thiopyridinium was removedfrom the protein conjugate by gel-filtration. All the derivatisationsteps were monitored using MALDI-TOF mass spectrometry.

[0106] Cell Uptake

[0107] Cell association was measured by flow cytometry using a FACScan(Becton Dickinson, USA). Free or vectorised compounds were incubatedwith K562 cells (5×10⁵ cells per ml) in Optimem medium at 37° C. forvarious periods of time.

[0108] Thereafter, the cells were washed twice and then resuspended in0.5 ml of ice-cold PBS for FACS analysis. Cell-associated fluorophoreswere excited at 488 nm and fluorescence was measured at 525 nm. Ahistogram of fluorescence intensity per cell (1×10⁴) was obtained andthe calculated mean of this distribution (Mean Fluorescence Intensity)was considered as representative of the amount of cell-associatedpeptide

[0109] In Vivo Model Systems

[0110] Mice (Balb/c) were immunized with either free or vectorisedantigens, and mixed in with each formulation was 15 μg of Heparin.Reagents were mixed and then used to immunize subcutaneously in eachflank. Mice were immunized on day 0 and after 3 weeks mice weresacrificed, spleens removed and single cell suspensions prepared. Threedays later, stimulated Balb/c derived LPS blasts (using dextran sulphateand LPS) were irradiated and incubated with the antigen. Spleen cells(6×10⁶/ml) and LPS blast (3×10⁶/ml) were incubated together for 7 days.Cytotoxic T cell activity was measured on day 6 using Cr⁵¹ labelled P815cells either unpulsed or pulsed with the antigen as targets. On day 7,remaining cells are restimulated with antigen pulsed P815 cells with theaddition of 20 U/ml of IL-2. All data has the non specific anti-P815cells CTL subtracted.

Example 1 Delivery of a Peptide Epitope

[0111] Compound Sequence Name (I) TYQRTRALV (SEQ ID NO:20) Flu NP (II)AWSFRVSYRGISYRRSR-TYQRTRALV SynB4/flu NP (SEQ ID NO:21) (III)RRLSYSRRRF-TYQRTRALV SynB3/flu NP (SEQ ID NO:22)

[0112] Cell Uptake

[0113] Flu NP epitope was conjugated to SynB3 and SynB4 vectors. First,we compared the cell uptake of free and conjugated flu NP. Flu NPepitope was conjugated to SynB3 and SynB4 vectors and labelled with afluorescent group (NBD). The compounds were incubated with K562 cellsfor various times and the cell uptake was measured using flow cytometry.

[0114] Incubation of the cells with free flu NP epitope resulted in avery low uptake as judged by the mean of fluorescence intensity (FIG.1). However, coupling the flu NP peptide antigen with either SynB3 orSynB4 vectors increased significantly its cell penetration. The cellpenetration was very rapid in the first 30 min and then plateauthereafter. The enhancement of internalisation was 2- to 5-folddepending on the vector used. At 45 min, the cell uptake of SynB4/flu NPwas about 5-fold higher than the one observed for free flu NP.

[0115] Immunization Studies

[0116] Balb/c mice (H-2d, female, 6-8 weeks) were immunized by theintradermal route (base of tail) with equimolar quantities of eitherfree or conjugated flu NP peptide. Specifically, each mouse received 25ug of flu NP peptide, either free, mixed with incomplete Freund'sadjuvant (IFA), or conjugated. Conjugated peptide groups also received15 ug of heparin. Spleens were harvested after three weeks. CTLresponses were measured using the ⁵¹Cr release assay with splenocytesthat had received two rounds of in vitro stimulation with free peptide.

[0117]FIG. 2 shows the results of the flu NP experiment. Specifically,naive mice as well as mice that had received flu NP peptide alone failedto elicit specific CTL responses (a and b, respectively). Two of threemice that were immunized with flu NP peptide in IFA elicited weak CTLresponses (c). In contrast, all mice that were immunized with eitherSynB3/flu NP (d) or SynB4/flu NP conjugates (e) elicited specific CTLresponses, with five of the six mice eliciting strong CTL responses.FIG. 2 shows that the responses elicited by the flu NP peptideconjugates are clearly stronger than those elicited by free peptide orfree peptide in IFA, a potent adjuvant.

Example 2 Delivery of a Recombinant Protein Antigen

[0118] Compound Sequence Name (IV) RDPV RDPV (V) AWSFRVSYRGISYRRSR-rDPVSynB4/rDPV ((SEQ ID NO:18)-rDPV) (VI) RRLSYSRRRF-rDPV SynB3/rDPV ((SEQID NO:11)-rDPV)

[0119] Cell Uptake

[0120] We also coupled rDPV protein with SynB3 and SynB4 vectors. Thecoupling procedure is described in the Protocols. First, we compared thecell uptake of free and coupled rDPV. The rDPV was labelled with afluorescent group (NBD) in order to measure its uptake. The compoundswere incubated with K562 cells for various times and the cell uptake wasmeasured using flow cytometry.

[0121] Incubation of the cells with free rDPV resulted in a very lowuptake as judged by the mean of fluorescence intensity (FIG. 3).However, coupling the rDPV protein with either SynB3 or SynB4 vectorsincreased significantly its cell penetration.

[0122] The enhancement of internalisation was 3- to 7-fold depending onthe vector used. As observed for the peptide epitope, SynB4 vectorenhanced rDPV cell penetration slightly better than SynB3. The celluptake of rDPV-SynB3 reached saturation faster than the one withrDPV-SynB4 (30 min versus 100 min).

[0123] Immunization Studies

[0124] C57Bl/6/c mice (female, 6-8 weeks) were immunized with 5 μg ofeither DPV protein, SynB3/rDPV conjugate, or SynB4/rDPV conjugate by theintradermal route (base of tail). The injection volume was 100 ul andalso contained 15 μg of heparin. Animals were immunized on day 0 and day21 with their spleens being harvested on day 42 for measurement of DPVspecific CTL responses. CTL responses were measured using the ⁵¹Crrelease assay with splenocytes that had received two rounds of in vitrostimulation.

[0125]FIG. 4 shows the mean CTL responses for this rDPV experiment.Naive mice (diamonds) failed to elicit specific CTL response (where aneffective response is defined as >10% specific lysis) while micereceiving rDPV protein alone elicited a weak CTL response (circles). Incontrast, mice that were immunized with either the SynB3/rDPV orSynB4/rDPV conjugate elicited strong CTL responses (inverted-trianglesand squares, respectively). FIG. 4 thus shows that the response elicitedby the rDPV conjugates are clearly stronger than that elicited by freeprotein.

1 22 1 13 PRT Artificial Sequence SITE (1, 13) Xaa may be either of anamino acid residue for which the side chain is non-polar, or an aminoacid residue for which the side chain is basic 1 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 2 5 PRT Artificial Sequence SITE (1)Xaa may be either of an amino acid residue for which the side chain isnon-polar, or an amino acid residue for which the side chain is basic 2Xaa Xaa Xaa Xaa Xaa 1 5 3 6 PRT Artificial Sequence SITE (1, 2, 4) Xaamay be either of an amino acid residue for which the side chain isnon-polar, or an amino acid residue for which the side chain is polar 3Xaa Xaa Xaa Xaa Xaa Xaa 1 5 4 18 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 4 Arg Xaa Xaa Arg Leu Xaa Tyr Xaa Xaa Xaa Xaa Phe Xaa Xaa XaaXaa 1 5 10 15 Xaa Arg 5 18 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 5 Arg Xaa Xaa Arg Leu Xaa Tyr Xaa Xaa Xaa Arg Phe Xaa Xaa XaaXaa 1 5 10 15 Gly Arg 6 18 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 6 Arg Xaa Xaa Arg Leu Xaa Tyr Xaa Arg Arg Arg Phe Xaa Xaa XaaXaa 1 5 10 15 Gly Arg 7 18 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 7 Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Arg Phe Ser Val SerVal 1 5 10 15 Gly Arg 8 18 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 8 Arg Ala Ala Arg Leu Ala Tyr Arg Leu Leu Arg Phe Ala Ile ArgVal 1 5 10 15 Gly Arg 9 18 PRT Artificial Sequence MOD_RES (9, 10) Nle 9Arg Ala Ala Arg Leu Gly Tyr Arg Xaa Xaa Arg Phe Gly Xaa Arg Val 1 5 1015 Gly Arg 10 18 PRT Artificial Sequence Description of ArtificialSequence Linear derivative of a beta-stranded antibiotic peptide 10 ArgGly Gly Arg Leu Ser Tyr Ser Arg Arg Arg Phe Ser Thr Ser Thr 1 5 10 15Gly Arg 11 10 PRT Artificial Sequence Description of Artificial SequenceLinear derivative of a beta-stranded antibiotic peptide 11 Arg Arg LeuSer Tyr Ser Arg Arg Arg Phe 1 5 10 12 17 PRT Artificial SequenceDescription of Artificial Sequence Linear derivative of a beta-strandedantibiotic peptide 12 Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Arg Xaa Xaa XaaXaa Xaa Xaa Xaa 1 5 10 15 Xaa 13 17 PRT Artificial Sequence SITE (1) Xaais Lys or Arg or Ala 13 Xaa Trp Xaa Phe Arg Xaa Xaa Tyr Arg Xaa Xaa XaaTyr Xaa Xaa Xaa 1 5 10 15 Xaa 14 17 PRT Artificial Sequence Descriptionof Artificial Sequence Linear derivative of a beta-stranded antibioticpeptide 14 Xaa Trp Xaa Phe Arg Val Xaa Tyr Arg Gly Ile Xaa Tyr Arg ArgArg 1 5 10 15 Xaa 15 17 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 15 Lys Trp Ser Phe Arg Val Ser Tyr Arg Gly Ile Ser Tyr Arg ArgSer 1 5 10 15 Arg 16 17 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 16 Arg Trp Ser Phe Arg Val Ser Tyr Arg Gly Ile Ser Tyr Arg ArgSer 1 5 10 15 Arg 17 17 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 17 Lys Trp Ala Phe Arg Val Ala Tyr Arg Gly Ile Arg Tyr Leu LeuArg 1 5 10 15 Leu 18 17 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 18 Ala Trp Ser Phe Arg Val Ser Tyr Arg Gly Ile Ser Tyr Arg ArgSer 1 5 10 15 Arg 19 17 PRT Artificial Sequence Description ofArtificial Sequence Linear derivative of a beta-stranded antibioticpeptide 19 Arg Ser Arg Arg Tyr Ser Ile Gly Arg Tyr Ser Val Arg Phe SerTrp 1 5 10 15 Ala 20 9 PRT Artificial Sequence Description of ArtificialSequence Flu NP 20 Thr Tyr Gln Arg Thr Arg Ala Leu Val 1 5 21 26 PRTArtificial Sequence Description of Artificial Sequence SynB4/flu NP 21Ala Trp Ser Phe Arg Val Ser Tyr Arg Gly Ile Ser Tyr Arg Arg Ser 1 5 1015 Arg Thr Tyr Gln Arg Thr Arg Ala Leu Val 20 25 22 19 PRT ArtificialSequence Description of Artificial Sequence SynB3/flu NP 22 Arg Arg LeuSer Tyr Ser Arg Arg Arg Phe Thr Tyr Gln Arg Thr Arg 1 5 10 15 Ala LeuVal

1. A conjugate of an antigen coupled to a linear derivative of aβ-stranded antibiotic peptide.
 2. A conjugate according to claim 1 wherethe linear derivative of a β-stranded antibiotic peptide is ofstructure: Nter-mid-Cter, in which mid is a peptide of formula (i):X1/3-(X1/2 or a bond)-(X or a bond)-X3-(X1 or a bond)-X1-X-X1/2-(X2/3 ora bond)-Db-(X2/3 or a bond)-X1/3  (SEQ ID NO:1);  where Db is eitherX3-X3 or X1-X1; and  wherein Nter is either an N-terminus orX1/X3-X1-X1/2-X1-X3  (SEQ ID NO:2); and  wherein Cter is either aC-terminus or X1/2-X1/2-X2/3-X1/2-X1-X3  (SEQ ID NO:3)  in which: eachX1, which may be identical or different, represents an amino acidresidue for which the side chain is non-polar; each X2, which may beidentical or different, represents an amino acid residue for which theside chain is polar; each X3, which may be identical or different,represents an amino acid residue for which the side chain is basic; X isany one of X1, X2 and X3;  wherein said peptide is linear and retainsthe ability to cross a mammalian membrane;  or a fragment thereofretaining the ability to cross a mammalian membrane.
 3. A conjugateaccording to claim 2 wherein X1 is selected from glycine, valine,norvaline, leucine, isoleucine, norleucine, proline, phenylalanine,methionine and tryptophan, X2 is selected from serine, threonine,tyrosine, asparagines and glutamine and X3 is selected from arginine andlysine.
 4. A conjugate according to claim 3 wherein said peptide is ofthe formula: R-(Xa)-R-L-X1/2-Y-X2/3-Db-(R or abond)-F-X1/2-X1/2-X2/3-X1/2-X1-R  (SEQ ID NO:4); wherein Xa is eitherX1-X1 or a bond.
 5. A conjugate according to claim 3 wherein saidpeptide is of the formula:R-(Xa)-R-L-(G/S/A)-Y-(R/S)-Db-R-F-(G/S/A)-X1-(R/S)-(V/T)-G-R  (SEQ IDNO:5).
 6. A conjugate according to claim 3 wherein said peptide is ofthe formula:R-(Xb)-R-L-(G/S/A)-Y-(R/S)-R-R-R-F-(G/S/A)-(T/I/V)-(R/S)-(V/T)-G-R  (SEQID NO:6) wherein Xb is either A-A, G-G, or a bond.
 7. A conjugateaccording to claim 3 wherein said peptide is of the formula selectedfrom the group: R-G-G-R-L-S-Y-S-R-R-R-F-S-V-S-V-G-R  (SEQ ID NO:7);R-A-A-R-L-A-Y-R-L-L-R-F-A-I-R-V-G-R  (SEQ ID NO:8);R-A-A-R-L-G-Y-R-_(n)L-_(n)L-R-F-G-Z-R-V-G-R  (SEQ ID NO: 9);R-G-G-R-L-S-Y-S-R-R-R-F-S-T-S-T-G-R  (SEQ ID NO:10); andR-R-L-S-Y-S-R-R-R-F  (SEQ ID NO:11). in which _(n)L is norleucine, and Zis norvaline.
 8. A conjugate according to claim 3 wherein said peptideis of the formula:X1/3-X1-X1/2-X1-R-X1-X1/2-X2-R-X1-X1-S/R-X2-Db-X2/3-X2/3  (SEQ IDNO:12).
 9. A conjugate according to claim 3 wherein said peptide is ofthe formula:(R/K/A)-W-(S/A)-F-R-X1-(S/A)-Y-R-X1-X1-(S/R)-Y-Db′-(R/S)-(R/L/_(n)L)  (SEQID NO:13) where Db′ is selected from L-L, _(n)L-_(n)L and R-R (where_(n)L is norleucine).
 10. A conjugate according to claim 3 wherein saidpeptide is of the formula:(R/K/A)-W-(S/A)-F-R-V-(S/A)-R-G-I-(S/R)-Y-R-R-R-(R/L)  (SEQ ID NO:14).11. The conjugate according to claim 3 wherein said peptide is of theformula selected from the group: K-W-S-F-R-V-S-Y-R-G-I-S-Y-R-R-S-R  (SEQID NO:15); R-W-S-F-R-V-S-Y-R-G-I-S-Y-R-R-S-R  (SEQ ID NO:16);K-W-A-F-R-V-A-Y-R-G-I-R-Y-L-L-R-L  (SEQ ID NO:17); andA-W-S-F-R-V-S-Y-R-G-I-S-Y-R-R-S-R  (SEQ ID NO:18).
 12. The conjugateaccording to any one of claims 1 to 11 wherein said antigen is selectedfrom the group consisting of a peptide, a whole protein, and a proteinsubunit.
 13. The conjugate according to claim 12 wherein the source ofsaid peptide, protein or protein subunit antigen is selected from thegroup consisting of a virus, a bacteria, an autologous protein and acancer protein.
 14. The conjugate according to claim 13 wherein saidvirus is selected from the group consisting of influenza virus;adenovirus; hepatitis A, B and C viruses; yellow fever virus; denguefever virus; HIV-1 and HIV-2; HSV1 and HSV2; Epstein-Barr virus;Retroperitoneal fibromatosis associated herpes virus, Human papillomavirus, Kaposi's sarcoma herpes virus, and cytomegalovirus (CMV).
 15. Theconjugate according to claim 13 wherein said bacteria is selected fromthe group consisting of Mycobacterium tuberculosis, Acne vulgaris,Propionibacterium acnes, Chlamydia trachomatis, Babesia microti,Ehrlichia risticii, Borrelia burgdorferi, Leishmania aethiopica, Candidaalbicans, Mycobacterium tuberculosis, Staphylococcus aureus,Staphylococcus pyogenes, Staphylococcus epidermis, Staphylococcussapropyticus, and Trypanosoma cruzi.
 16. The conjugate of claim 13wherein said cancer is selected from the group consisting of Acutemyelogenous leukaemia (AML), Acute lymphocytic leukaemia (ALL), Chronicmyelogenous leukaemia (CML), Chronic lymphocytic leukemia (CLL), Hairycell leukemia, Myeloma, and all solid tumors of all tissue types.
 17. Amethod of enhancing the immune response of a mammal to an antigen whichcomprises administering to the mammal the conjugate of any one of claims1 or
 3. 18. A composition comprising the conjugate of any one of claims1 or 3 in a pharmaceutically acceptable carrier.
 19. A method ofpreparing the conjugate of any one of claims 1 or 3 comprising the stepsof: (a) expressing in a host cell a nucleic acid sequence encoding saidconjugate and (b) recovering said conjugate from said host cell.